Fig. 2.
ATM kinase activity is essential for efficient signal join formation during chromosomal V(D)J recombination of DNA-PK–deficient v-abl–transformed pro-B cells. (A) Diagram of the pMX-DELSJ substrate used in chromosomal V(D)J recombination assays. The diagram is adapted from Bredemeyer et al. (27). The pMX- DELSJ vector has a single pair of RSs. Normal V(D)J recombination between the two RSs leads to excision of the intervening sequences and formation of a chromosomal SJ. Also shown is a schematic representation of unrearranged (UR) cleavage intermediates (3′ SE) and joining products (SJ) of pMX-DELSJ. The long terminal repeats (LTR), IRES-hCD4 cDNA (hCD4), 5′ 12-RS and 3′ 23-RS (filled and open triangles, respectively), EcoRV (EV) site, and C4 probe are indicated. (B) Southern blot analyses of the rearrangement status of pMX-DELSJ V(D)J recombination substrate (A) in two pairs (DELSJ4 and DELSJ6) of DNA-PKcs−/−ATMC/C, DNAPKcs−/−ATM−/−, and XRCC4−/− control v-Abl–transformed pro-B cells. DNA was digested with EcoRV and probed with the indicated C4 probe. The UR band is 5 kb, the SJ band is 4 kb, and the 3′SE band is 2.2 kb. (C) Southern blot analyses of the rearrangement status of pMX-DELSJ V(D)J recombination substrate (A) in DNA-PKcs−/− cells with or without treatment of ATM inhibitor (15 μM). (D) Fidelity analysis of the chromosomal signal joints recovered from DNA-PKcs−/−ATMC/C and PKcs−/−ATM−/− cells with pMX-DELSJ substrates.